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Abstract

We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a hydroquinone dioxygenase, YaiA, described here, and a hydroquinone reductase, YaiB, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the yahCD-yaiAB operon. The operon is induced by copper to prevent the synergistic toxicity of quinones and copper. The hydroquinone dioxygenase, YaiA, converts hydroquinones to 4-hydroxymuconic semialdehyde, using molecular oxygen as oxidant according to the reaction: hydroquinone + O2 → 4-hydroxymuconic semialdehyde + H+ We here describe two methods for measurements for hydroquinone dioxygenase activity, based on oxygen consumption measured with an oxygen electrode and the spectrophotometric detection of 4-hydroxymuconic semialdehyde. Both assays are conducted with crude cell extracts.

Preparation of crude E. coli extracts containing YaiAE. coli BL21(DE) containing plasmid pCA31 (Mancini et al., 2015) was grown aerobically in 300 ml of LB broth containing 100 μg/ml of ampicillin at 37 °C to an OD at 600 nm of 0.4. Then, 0.1 mM isopropyl-β-D-thiogalactoside was added, and growth was continued for 8 h at room temperature. Cells were harvested by centrifugation, and pellets suspended in 20 mM Tris-Cl (pH 7.5), containing 1 mM 4-hydroxybenzoate to stabilize protein as described elsewhere (Moonen et al., 2008), 1 mM phenylmethylsulfonyl fluoride, and 100 μg/ml of DNaseI. Cells were then broken by three passages through a French Press at 40 MPa, and cell debris was removed by centrifugation at 4 °C for 20 min at 90,000 x g. The supernatant was filtered through a 0.45 μm nitrocellulose filter and stored frozen at -20 °C until used.

YaiA Activity assay by spectrophotometry

Mix 50 mM Tris-Cl, pH 7.5, 10% (v/v) glycerol with 1 mg of crude extract
in a total volume of 990 µl and warm to 30 °C in the spectrophotometer.
This temperature was chosen since the origin of YaiA is L. lactis,
which grows best at 30 °C.

Zero the spectrophotometer and record
the baseline spectrum from 300 to 400 nm. Start the reaction by adding
10 µl of 100 mM hydroquinone (final concentration 1 mM) and record
spectra every 5 to 15 min, depending on the rate of the reaction.
Calculate the specific activity from the increase in absorption at 320
nm, using an extinction coefficient for 4-hydroxymuconic semialdehyde of
11,000 M-1 cm-1 (Moonen et al., 2008; Spain and Gibson, 1991). Running
spectra rather than measuring absorption at 320 nm is important to
assured that the desired reaction is measured in the crude extracts.
This should be further validated by calculating difference spectra (see
Figure 1 and Note 1).

Enzyme kinetics

To determine the Km for hydroquinone, run enzyme reactions with 1, 3, 10, 30, 100, 300 and 1,000 µM hydroquinone.

Determine the initial reaction rate, v0, for each reaction. If the
reaction is linear, v0 is the slope of the linear regression of the data
(e.g. calculated with the corresponding function built into Excel). If
the reaction curve is sigmoidal, only the initial (highest) slope should
be considered. Plot v0versus the substrate concentrations, S.

Fit the curve to obtain the affinity for the substrate, Km, and the
maximal velocity, vmax. Alternatively, plot v0versus 1/S to obtain a
Lineweaver-Burk plot. The regression line intercepts the ordinate at
1/vmax and the abscissa at -1/Km.

When a
constant rate of oxygen consumption is attained (basal oxygen
consumption due to other enzyme activities), inject 10 µl of 100 mM
hydroquinone into the chamber and continue to record the oxygen
consumption.

Subtract the basal oxygen consumption from the rate
observed with hydroquinone. Calculate the specific activity by
calculating the oxygen consumption, based on the chamber volume and the
oxygen content of the buffer, which can be taken from Estabrook (1967)
or, if high precision is required calculated as described by Rasmussen
and Rasmussen (2003). Under our conditions, buffer at 30 °C contains 222
µM O2.

Enzyme kinetics

To determine the Km for hydroquinone, run enzyme reactions with 1, 3, 10, 30, 100, 300 and 1,000 µM quinone.

Determine the initial reaction rate, v0, for each reaction and plot v0versus the substrate concentrations, S.

Fit the curve to obtain the affinity for the substrate, Km, and the
maximal velocity, vmax. Alternatively, plot 1/v0versus 1/S to obtain a
Lineweaver-Burk plot, from which Km and vmax can be determined.

Representative data

Figure 1. Absorption spectra of hydroquinone conversion to 4-hydroxymuconic semialdehyde. The lower curve was recorded at time 0 and the upper curve after 60 min of reaction. Inset, difference spectrum recorded between YaiA-containing extract incubated with and without 1 mM hydroquinone for 1 h, showing the absorption maximum of the reaction product at 320 nm. Axis labels are the same as in the main figure. Note that the spectra will look differently if substituted hydroquinones are used as the substrate for YaiA, but are not expected to change for different YaiA-like enzymes.

Figure 2.Oxygen consumption by hydroquinone dioxygenase activity in crude extracts. The reactions were induced by the addition of 1 mM hydroquinone at 2.5 min. Curves from top to bottom are: 1 mg of cytoplasmic extract from E. coli not expressing hydroquinone dioxygenase, extract heated to 95 °C for 5 min, and 1 mg of cytoplasmic extract from E. coli strain expressing YaiA of Lactococcus lactis from a plasmid.

Notes

If difference spectra between unreacted and reacted extracts show the expected absorption spectrum for 4-hydroxymuconic semialdehyde, activity may be measured by continuous recording at 320 nm.

Hydroquinone dioxygenase from L. lactis could not be purified in an active state and in crude cell lysates of L. lactis, the activity was too low to be detected.

4-hydroxymuconic semialdehyde is very labile and difficult to isolate and characterize. However, no change in absorbance was observed over at least one hour once the reaction had come to completion.

Hydroquinone must be prepared fresh on the day of use due to the spontaneous auto-oxidation to benzoquinone in the presence of oxygen. No significant autoxidation of the stock solution was observed within 8 h.

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